Predicting resistance of sheep to Haemonchus contortus infections

Veterinary Parasitology, 5 (1979) 365--378 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands

365

PREDICTING RESISTANCE OF SHEEP TO HAEMONCHUS C O N T O R T U S INFECTIONS

GEORGE G. R I F F K I N * and COLIN DOBSON Department o f Parasitology, University o f Queensland, Brisbane, Queensland 4067 (Australia)

*Present address: Regional Veterinary Laboratory, Hamilton, Victoria 3300 (Australia) (Accepted 12 February 1979)

ABSTRACT Riffkin, G.G. and Dobson, C., 1979. Predicting resistance of sheep to Haemonehus contortus infections. Vet. Parasitol., 5: 365--378. In vitro cultures of lymphocytes from worm-free sheep responded to larval and adult H. contortus antigens by undergoing blast transformation. The level of response varied considerably between animals but was heritable and positively correlated with resistance to subsequent primary, secondary and trickle infection by the parasite, but not with age of the sheep older than 5 months. L y m p h o c y t e responses to phytohaemagglutinin and H. contortus antigens were not present at birth in lambs but developed during the first 5 months in the absence of infection. The proportion of the infecting dose of larvae which was inhibited at the early fourth stage correlated with age o f the sheep and with the increase in lymphocyte response to H. contortus antigens during primary infections with H. contortus. Sheep which were most susceptible to infection had the lowest lymphocyte responses to H. contortus antigens but had the highest rate of weight gain during infection. It is suggested that man has selected the most productive animals at the expense of resistance to H. contortus. The definition of genetically controlled markers on lymphocytes is seen as a means by which disease resistant strains of animals may be selected.

INTRODUCTION

A major goal of agriculture is to increase productivity. However, the selection of highly productive plant and animal genotypes has often resulted in serious reductions of resistance to their natural parasites (Day, 1974). High stocking densities of ruminants on highly productive pastures in areas with humid temperate or sub-tropical climates also favour the survival and transmission of nematode parasites. Haemonchus contortus is a trichostrongylid parasite of sheep which causes great economic loss, but all sheep are not equally affected by the parasite nor do different strains of H. contortus have the same pathogenicity (Allen et al., 1958; Whitlock, 1958; Conway and Whitlock, 1965). It has been demonstrated that sheep which have haemoglobin type A and those which synthesise haemoglobin rapidly may be able to withstand the pathogenic effects o f / / . contortus better than sheep which do not have

366

these characteristics (Bemrick et al., 1958; Evans and Whitlock, 1964). However, sheep which better resist establishment of H. contortus by virtue of their immunological competence may not necessarily be able to overcome the pathogenic effects of parasites which survive these immunological reactions. Resistance to infection therefore has different meanings in terms of the parameter being measured. The ability of sheep to withstand the pathogenic effects of H. contortus depends upon homeostatic mechanisms and is better termed resilience. On the other hand, resistance implies an antagonistic response acting against the development and continued presence of the parasite, is defined by the immunological competence of the host and may be manifested by failure of the parasite to become established, inhibition of development through the parasitic larval stages, reduced fecundity of adults and elimination of existing infections as in the classical 'self-cure' reaction. These manifestations arise from a combination of innate resistance and immunity which is acquired as a result of the increasing age of the host or its previous experience with the parasite. This paper records experiments which were designed to investigate the relationship between responses of sheep lymphocytes when cultured in vitro with H. contortus antigens, and the resistance of the sheep to infection with the parasite. MATERIALS AND METHODS Animals and experimental procedures

In a series of five experiments, 26 Merino × Border Leicester sheep, reared worm-free and ranging in age from 5 months to 18 months, were infected with 12,000 L3 Haemonchus contortus. Eight further 8-month-old sheep were infected and reinfected 8 weeks later with similar doses of H. contortus larvae. Trickle infections were established in five 5-month-old sheep by administering 500 L3 H. contortus daily for 35 days. The final experiments compared the in vitro lymphocyte responses of five worm-free ewes with six of their 5-monthold worm-free lambs to antigens prepared from H. contortus. All the sheep were the progeny of one ram. Parasite

The H. contortus larvae were obtained from 7-day-old faeces cultured from sheep infected with the local field strain of H. contortus. The larvae were aged in water at 4°C for 14 days before being administered to sheep in 2 ml water on the back of the tongue and washed down with 20 ml water. Antigens

Antigens were prepared by disintegrating either third-stage (L3) or adult (Ad) H. contortus, which had been thoroughly cleaned by repeated washing in

367

0.15 M saline. Approximately 5 ml packed worms were homogenized with a minimum amount of 0.15 M saline in a glass homogenizer. The homogenate was clarified by centrifugation and the antigen supernatant sterilized by repeated filtration through 0.2 pm Millipore membranes. The protein concentration was measured on each antigen batch by the Biuret reaction and adjusted to 2 mg/ml protein with sterile RPMI 1640 (Grand Island Biologicals, New York, N.Y.).

In vitro cultivation of ovine whole blood A modification of the method developed by Han and Pauly (1972) was used. Ten-ml sterileblood samples were taken from the jugular vein of sheep and from uninfected controls 7 days, 2 days and 10 rain before infection as well as every other day after infection.The samples were dispensed in E D T A , clotted for serum or diluted 1 : 30 with R P M I 1640 medium with 2 % heat decomplemented foetal calf serum (FCS) (Medos, Brisbane) and 0.03% preservative-freeheparin. The diluted blood samples were dispensed in 2-ml aliquots into 15 X 115-mm sterileglass tubes and tightly stoppered with neoprene bungs. Triplicatecontrol tubes, together with triplicatestreated with 10 pg phytohaemagglutinin-P (PHA) Difco, Detroit, U.S.A.) in 20 pl thirdstage larval(La) or adult (Ad) antigen (containing 200 pg parasiteprotein) were incubated for 120 h at 37°C. All cultures were vortex mixed at 12-h intervals and pulsed with I pCi tritiatedthymidine (allTdr, S A 2 Ci/mM) (Radiochemical Centre, Amersham) 12 h before harvesting. Cultures were stopped by adding 2 drops Zapoglobin (Coulter Electronics) which also lysed the erythrocytes. The remaining cellswere filteredonto glass-fibrepads (Whatman GFC), filterwashed with 30 ml saline,bleached with 2 ml hydrogen peroxide and dehydrated with 30 ml methanol. The pads were air dried in pill-tubes(ACI, Sydney, Australia) immersed in 2 ml toluene based fluor conraining 6 g P P O (2,5-diphenyloxazole) and 0.2 g P O P O P [1,4-bis(2-{5-phenyloxazolyl ))-benzene,phenyl-oxazolylphenyl-oxazolyl-phenyl] in I I toluene, stoppered, placed in Beckman glass scintillationvials,and/3 particleemission measured in a Beckman LS 100C liquid scintillationcounter with external standard ratio.Counts were converted to disintegrationsper minute (dpm) and the mean d p m PHA, or antigen stimulated cultures divided by the mean control d p m was expressed as a ratio.This ratio,the stimulation index (SI) was taken to be a measure of the immunological response of the cultured lymphocytes to each mitogen. Parasitological techniques Faecal egg counts were done by the modified McMaster method every other day on each animal 18 days after infection (Mines, 1977). Twenty-three sheep from the first series of experiments, together with five sheep from the trickle experiments and six reinfected sheep were killed 35 days after infection or re-

368

infection. Abomasal parasites were recovered from washings and from 1% HCl-pepsin digests of the abomasal mucosae. H. contortus were identified and counted as early fourth-, late fourth- or fifth-stage larvae and mature adult parasites. Haemoglobin types

Haemoglobin from washed lysed sheep erythrocytes was electrophoresed in starch gel together with known A, B and AB ovine haemoglobin standards. Mathematics

Analyses of variance, correlation coefficients and covariance were done using standard programmes from the Department of Animal Production and the Computer Centre, University of Queensland. RESULTS

Preliminary experiments established the o p t i m u m conditions for the in vitro cultivation of sheep whole blood outlined above. They showed that lymphocyte responsiveness to PHA and H. contortus antigens was not present at birth in lambs but developed during the first 5 months of life in the absence of infection. The pre-infection responsiveness of the cultured peripheral blood lymphocytes to H. contortus antigens varied between sheep and appeared to relate inversely to faecal egg counts during subsequent H. contortus infections.

¢ 'T'I 16 nfected

== 12

i

Jt

E

•.~

0

I

0

i

i

10

I

I

20

I

i

I

30

I

40

I

i

50

I

I

i

60

Days after infection

Fig. 1. The kinetics of l y m p h o c y t e responses (stimulation indices) of w h o l e b l o o d cultures f r o m three c o n t r o l ( . . . . . ) and three sheep i n f e c t e d w i t h 12,000 L 3 H. contortus to H. contortus (H.c.) larval (o) a n d a d u l t ( × ) antigens.

369

In vitro lymphocyte responses to both third-stage larval (L3) and adult (Ad) H. contortus antigens rose to a peak during the third week of primary infections and declined thereafter, but remained elevated compared with the responses of lymphocytes from non-infected control sheep {Fig. 1). Primary Haemonchus contortus infections

In the first series of experiments the pre-infection lymphocyte stimulation indices (SI) of 26 sheep to be infected with H. contortus were measured twice weekly for 3 weeks. The sheep were ranked by the level of their average preinfection lymphocyte response to L3 or Ad H. contortus antigen. The top 13 animals were designated high responders (HR) (SI, L3 3.4 + 0.4, Ad 2.8 -+ 0.4); the bottom 13, low responders (LR) (SI, L3 1.1 + 0.05, Ad 1.1 + 0.05). Sheep in the HR group had longer prepatent periods, voided fewer parasite eggs, and fewer H. contortus were recovered 35 days after infection than from LR sheep (Table I). Faecal egg counts increased more rapidly to higher levels in LR than HR sheep (Fig. 3). The sheep were also ranked as resistant or susceptible according to the percentage of the infecting dose which was established in the abomasum at slaughter, the prepatent period of the infection and the mean faecal egg count between days 28 and 35. Overall the mean values for resistant or susceptible groups for each parameter closely paralleled the values for the HR and LR groups (Table I). The mean pre-infection whole blood culture stimulation indices for both L3 and Ad antigens correlated negatively with the total recovery of H. contortus, the total numbers of eggs voided in the faeces between days 28 and 35 (Fig. 2) and with weight gain throughout the experiment (Tables I and II). Non-infected high- and low-responder animals retained their lymphocyte SI levels for the duration of the experiment and the pre-infection lymphocyte stimulation indices for all the animals older than 5 months were not related to either age or sex of the host (Fig. 1, Table II). In vitro whole blood cultures from older sheep responded more strongly to L3 antigen after infection than did those from younger animals, and older sheep harboured fewer adult parasites (Table II). As would be expected, the mature sheep gained less weight during the experiment than did the growing lambs. There was a negative correlation between pre-infection SI and weight gain during the infection but there was no significant correlation between the age of the sheep and their pre-infection L3 antigen SI. Moreover, older sheep inhibited the development of more H. contortus at early fourth stage than did lambs (Table II). This correlated with an increase in SI during infection rather than with the pre-infection lymphocyte SI to H. contortus antigens. Secondary Haemonchus contortus infections

HR sheep (pre-infection SI to L3 antigen 8.0 -+ 1.5, Ad 5.2 + 1.6) reinfected with 12,000 larvae 56 days after a primary infection with a similar dose of

Percent H.c. 11.6 establishment Percent dose 8.5 adult parasites Prepatent 24.5 period (days) Mean faecal egg 3324 count (days 28--35) Weight before 30.5 infection (kg) Weight gain (kg) 3.8 Pre-infection 10.7 haemoglobin level (g%) 13

1.5

11 11 11

3.4

0.4 0.3

13

10

2.5

1561

12

1.9

4.5 10.9

24.8

899

25.5

5.4

10.1

L3 antigen lymphocyte stimulation index ranking

1.3

1.4

1.5

0.5 0.2

2.4

237

SE

x

n

x

SE

Observed values resistant 2 sheep

Predicted HR sheep 1

12 12

12

13

13

10

12

n

20.9

27.3

28.7

5.0 11.6

23.0

11377

x

0.7

5.9

4.6

0.3 0.3

3.3

3180

SE

Predicted LR 1 sheep

12 12

13

13

12

9

11

n

19.8

30.7

30.3

4.4 11.6

22.4

13801

x

0.21

4.6

4.0

0.3 0.3

2.6

2875

SE

Observed values susceptible 2 sheep

10 10

10

13

12

9

11

n

Levels of infection established in high (HR) and low (LR) responder groups of sheep 35 days after primary infection with 12,000 L 3 Haemonchus contortus (H.c.) compared with observed levels of infection in resistant and susceptible sheep from the HR and LR groups

TABLE I

--3 Q

1.5

24.8

593

2.5

8.7

3.4

0.4 0.3

30.5

3.8 10.7

1182

1.9

11.7

11 11

11

13

13

10

12

4.5 10.9

24.8

899

25.5

5.4

10.1

0.5 0.2

2.4

273

1.3

1.4

1.5

12 12

12

13

13

10

12

5.0 11.6

23.0

12818

20.7

27.1

28.7

0.4 0.3

3.3

3150

0.6

5.9

4.6

11 11

13

13

12

9

11

4.4 11.6

22.4

13801

19.8

30.7

30.3

0.3 0.3

2.6

2875

0.21

4.6

4.0

10 10

10

13

12

9

11

1,2Sheep in 1 ranked by pre-infection lymphocyte transformation ratio. The same sheep were ranked in 2 according to the percent Haemonchus contortus established, n varies depending on availabilityof data.

Weight before infection (kg) Weight gain (kg) Pre-infection haemoglobin level (g%)

28-35)

Mean faecal egg count (days

Percent H.c. establishment Percent dose adult parasites prepatent period (days)

Ad antigen lymphocyte stimulation index ranking

"--1 t-t

372 TABLE II Correlation coefficients (r) between host and parasite parameters for sheep infected with 12,000 Haemonchus contortus larvae n1

r

p2

Pre-infection in vitro adult (Ad) H. contortus lymphocyte stimulation index ($I) o f sheep compared with:-

Total recovery of H. contortus (day 35) Total worm eggs voided in faeces (days 28--35) Weight gain of sheep between days 0 and 35 following infection Age of sheep at day 0

24

--0.51

~0.02

24

--0.45

~0.05

11 34

--0.76 0.02

~0.01 N.S.

0.39 0.51 0.10 0.58

~0.05 ~0.02 N.S. ~0.01

28

0.58

~0.01

17 19

0.54 0.09

~0.02 N.S.

Age o f sheep at day 0 o f infection correlated with:-

Mean larval H. contortus antigen (L3) l y m p h o c y t e SI, for days 14--28 minus pre-infection SI Total adult parasites recovered Pre-infection L3 antigen SI Percent larval inhibition

28 21 30 28

Percent H. contortus larvae inhibited correlated with:-

Age of sheep at day 0 Mean L3 antigen l y m p h o c y t e antigen SI for days 14--28 minus pre-infection SI Pre-infection L3 antigen l y m p h o c y t e SE

I n, number of paired data. 2p, probability value.

_~

10

!:o ~.~

8

I

_~ !.~ --2:1

•

oO

,

"o.

• O.

:~

•

• "

oil

Oo

,.

o

•

OOo

o" O °

o°

• •

0 10 20 30 40 50 Mean H.contortus eggs per gram faeces X10-3(days 28 -35~'O-'a'~nd percent worm establishment day 35 •

Fig. 2. The relationship between pre-infection in vitro lymphocyte stimulation indices induced by larval (L3) Haemonchus contortus antigen and subsequent resistance of sheep to infection with 12,000 H. contortus larvae.

373

H. contortus v o i d e d f e w e r eggs, and h a r b o u r e d f e w e r parasites t h a n r e i n f e c t e d L R sheep ( p r e - i n f e c t i o n SI t o L3 antigen 3.2 + 0.9, A d 2.6 + 0.5). T h e r e was n o d i f f e r e n c e b e t w e e n t h e groups in t h e n u m b e r o f i n h i b i t e d larvae r e c o v e r e d (Table III). T h e residual faecal egg c o u n t f r o m t h e p r i m a r y i n f e c t i o n rose following r e i n f e c t i o n in b o t h L R and H R sheep. 'Self c u r e ' was h o w e v e r initiated s o o n e r and m o r e e f f i c i e n t l y in H R t h a n L R sheep. F e w e r parasite eggs were v o i d e d t h e r e a f t e r b y H R c o m p a r e d with L R sheep (Fig. 3). TABLE III Levels of infection established in high (HR) and low (LR) responder groups of sheep 35 days after reinfection with Haemonchus contortus (H.c.) (12,000 larvae 56 days after primary infection with 12,000 larvae) compared with observed levels of infection in resistant and susceptible sheep from the HR and LR groups L3, A d I antigen SI Observed values L3, Ad antigen SI Observed values predicted H R sheep resistant sheep predicted LR sheep susceptible sheep ~2

SE

n

~

SE

n

SE

n

~

SE

r/

NumberH.c. 808 171 2 934 160 3 2135 513 4 2447 573 3 established Number H.c. 102 102 2 35 22 3 728 400 4 1003 418 3 adult NumberH.c. 705 273 2 841 208 3 1630 442 4 1801 576 3 inhibited larvae Mean faecal 1000 730 4 857 645 4 3943 686 4 4124 564 4 egg count 1 L3 (larval) and Ad (adult) antigen lymphocyte SI (stimulation index) rankings were the same. 2£, mean; SE, standard error; n, number, only two of the predicted HR group of sheep were killed.

Trickle H a e m o n c h u s c o n t o r t u s infections Five lambs w e r e r a n k e d a c c o r d i n g t o t h e i r p r e - i n f e c t i o n l y m p h o c y t e SI t o L3 and Ad H. contortus antigens ( H R g r o u p n = 2, SI t o L3 antigen 3.9 + 0.1, and t o Ad antigen 3.0 + 0.3; L R group n = 3, SI t o L3 2.4 + 0.3, Ad 1.5 + 0.2). T h e H R animals v o i d e d f e w e r parasite eggs and had f e w e r parasites established in t h e m with f e w e r a d u l t w o r m s t h a n t h e L R lambs. T h e r e was n o d i f f e r e n c e in p r e p a t e n t p e r i o d b e t w e e n t h e groups (Fig. 4, T a b l e IV).

Inheritance o f pre-infection in vitro l y m p h o c y t e stimulation index to La and A d H a e m o n c h u s c o n t o r t u s antigen In t h e final e x p e r i m e n t t h e in vitro p r e - i n f e c t i o n l y m p h o c y t e responses o f five ewes t o H. contortus antigens were c o m p a r e d w i t h t h o s e o f t h e i r six lambs w h e n t h e y w e r e 5 m o n t h s o f age. T h e p r e - i n f e c t i o n l y m p h o c y t e re-

374

r-I I I l

lO X

self.cure E ~6

15

7

o X

I I

TI

,

E

I

10

ii'

~t o

l

10 Infected

20

3o

40

20

2.5

Days a f t e r infection

I

30

35

Days after infection

Fig. 3. Progressive mean faecal egg counts for high (% • ) and low (no symbol) responder sheep during primary ( . . . . . ) and secondary ( ) infections with 12,000 H a e m o n c h u s cont o r t u s larvae. Sheep classified as high and low responders according to their pre-infection in vitro lymphocyte stimulation index to H. c o n t o r t u s antigen. The graph shows the time of infection as zero days for both primary and secondary infections. Fig. 4. Progressive mean faecal egg counts for two high (o) and three low (e) responder sheep during trickle infections with 500 H a e m o n c h u s c o n t o r t u s larvae given daily (days 0--35). Sheep classified as high and low responders according to their pre-infection in vitro lymphocyte stimulation index to adult H. c o n t o r t u s antigen.

TABLE IV Levels of infection established in two high (HR) and three low (LR) responder sheep after 35 daily infections with 500 H a e m o n c h u s c o n t o r t u s (H.c.) larvae compared with observed levels of infection in resistant and susceptible sheep from the HR and LR groups L~, Ad I antigen SI predicted HR sheep

Number H.c. established 1080 Number H.c. adult 60 Number H.c. inhibited larvae 1018 Prepatent period 21 Mean feacal egg count (days 28-35) 225

L3, Ad antigen SI predicted LR sheep

SE

n

~

SE

n

319

2

2143

323

3

50

2

572

114

3

267 1

2 2

1881 20

109 0.7

3 3

125

2

1014

406

3

1L~ (larval) and Ad (adult) antigen lymphocyte SI (stimulation index) rankings were the same. Predicted and observed resistance rankings were also the same.

375 sponses of ewes to L3 H. contortus antigen correlated significantly with those of their lambs (r = 0.84, P = 0.02). There was no correlation between the preinfection l y m p h o c y t e responses of ewes and lambs to Ad H. contortus antigen (r = 0.3, P NS) (Table V). T~LEV

The pre-infection in vitro lymphocyte stimulation indices of five ewes compared with those of their 5-month-old lambs to phytohaemagglutinin (PHA) and larval (L3) and adult (Ad)

Haemonchus contortus antigens Lymphocyte stimulation index Ewe Lamb No.

31 33 33 38 42 86

Mitogen

No.

PHA

L~

Ad

17 24 24 58 9 77

1.2 2.7 2.7 4.8 1.5 2.8

1.4 2.3 2.3 4.0 1.3 3.4

32 34 35 39 45 12

Mitogen

PHA

L3

Ad

9 33 9 4 35 72

0.7 0.8 0.9 1.0 0.7 1.1

0.9 1.1 1.3 1.2 1.2 1.1

Analyses o f covariance Analyses of covariance on data from the first series of experiments showed that pre-infection l y m p h o c y t e SI levels gave the best single prediction of the potential of sheep to resist H. contortus. Sheep with low haemoglobin levels together with high body weights gave the best combined prediction independent of the pre-infection l y m p h o c y t e SI to H. contortus antigens (df = 20, r = 0.67, P-- 0.01) (Table I).

Influence o f ovine haemoglobin type on resistance to Haemonchus contortus Most sheep in the study had type A or AB haemoglobin, few were type B (ratio 3 : 3 : 1). There was no detectable relationship between the numbers of faecal eggs voided by or the percent establishment of H. contortus and haemoglobin type but only six sheep were studied of which only one was type B. Type A haemoglobin sheep had elevated haemoglobin levels and greater pre-infection l y m p h o c y t e stimulation indices to both L3 and Ad H. contortus antigens when compared with type B sheep (Table VI).

376 TABLE VI Relationships between ovine haemoglobin type, haemoglobin levels, pre-infection ovine lymphocyte responses to L 3 and Ad H. c o n t o r t u s antigens and H. c o n t o r t u s infections Sheep

Mean values (+- standard error)

haemoglobin type

Percent parasite establishment

Mean faecal egg

Pre-infection levels

count (× l f f 3 )

Haemoglobin Lymphocyte SI ~ L 3 antigen Ad antigen

A AB B

10.0 (+2.6) 32 10.6 (+5.6) 2 11.7 1

2.8 1 0.3 1 2.0 1

11.4 (+2.8) 14 11.6 (±0.3) 11 10.9 (±1.0) 4

2.8 (+-0.4) 2.3 (±0.3) 14 14 1.8 (±0.4) 1.5 (+-0.3) 11 11 1.4 (+0.2) 1.4 (±0.2) 4 4

1SI stimulation index. 2 Number.

DISCUSSION

Whitlock and Madsen (1958) investigated the feasibility of genetic selection for resistance to trichostrongyloidosis using progeny from two rams. They concluded that the genes for resistance and resilience were n o t necessarily associated with each other and that the genetic relationship was complex although it was highly heritable. They stated that it was difficult to determine a true p h e n o t y p e in a situation in which the expression of the character was subject to variation. Inability to detect resistant animals w i t h o u t subjecting them to controlled experimental infections has been a handicap to the selection of H. contortus resistant sheep. Jilek and Bradley (1969) demonstrated that differences in resistance to H. contortus b e t w e e n individual sheep within Rambouillet and Florida native flocks were related to haemoglobin type b u t that the greater differences in resistance which existed between the breeds could n o t be explained on the basis of haemoglobin type. No correlation was demonstrated between haemoglobin t y p e and resistance to H. contortus in the present work, b u t very few sheep were investigated and most of these animals had t y p e A haemoglobin. T y p e A b u t n o t t y p e B haemoglobin correlated with elevated haemoglobin levels; this was also characteristic of the data of Evans and Whitlock (1964). Moreover t y p e A sheep had greater pre-infection l y m p h o c y t e stimulation indices than other sheep. Resistance of sheep to experimental H. contortus infections related to l y m p h o c y t e responsiveness to antigens from this parasite and was inherited in a predictable manner. This was shown b y the significant correlations between the pre-infection responsiveness of l y m p h o c y t e s from ewes and lambs to H.

377 contortus larval antigens. Lymphocyte responsiveness to H. contortus develops

early in the life of sheep in the absence of the parasite possibly as a genetically controlled response to heterophile antigens. Immunization of these sheep by previous infection was ruled out because the flock had been maintained wormfree for five generations; faecal egg counts were consistently negative although small numbers of coccidial oocysts were occasionally found. It is interesting that the inheritance of lymphocyte responsiveness to H. contortus between ewes and their lambs was significant only for L3 and not for Ad H. contortus antigen. This suggests that the trait may have evolved with respect to larval establishment rather than the development of the parasite. Moreover older sheep inhibited the development of more H. contortus at the early fourth stage than lambs, although the age of the sheep did not influence the numbers of larval H. contortus established. Thus inhibition of larval development appeared to be independent of the inherited level of immunological responsiveness to H. contortus antigens; rather, it was associated with the efficiency with which sheep mobilized their immune response to these antigens, which increased with their age. In view of the possibility that the increased immunological competence of sheep compared with lambs may have masked the inherited immunological trait demonstrated during primary pulse infections with 12,000 H. contortus, sheep were ranked and infected as before and then reinfected. Additionally, sheep with daily trickle infections were studied since this may more closely parallel natural infections. In each case the HR group of sheep voided fewer eggs and harboured fewer parasites compared with the LR animals. These experiments confirmed that we were observing manifestations of intrinsic differences of lymphocyte responsiveness related to the genetic structure of sheep. The increase in immunological competence which resulted from challenge and trickle infections reinforced the differences in lymphocyte responsiveness between HR and LR sheep. This was reflected by the earlier onset of a more efficient 'self,cure' reaction in HE compared with LR sheep. Overall our data indicate that sheep with high rates of weight gain may be less able to react against H. contortus antigens and are therefore less resistant to infection with this parasite (Table I). The implication arises that, while 'man-guided' evolution has increased the potential of sheep to produce meat and wool, selection has produced animals which are less resistant to H. contortus. It is possible that the inverse relationship demonstrated here between weight gain and resistance to H. contortus may not be absolute. For example, the resilience of individual sheep to overcome the pathogenic effects of H. contortus may not be linked with their immune response. Analyses of covariance on combined data suggest that the inherent immune response and increased immunological competence in older sheep may operate by different mechanisms. Resistance to the establishment and fecundity of the parasite relates to genetically controlled lymphocyte responsiveness in sheep. On the other hand, age resistance, which also influences the degree of larval inhibition, is associated with haemoglobin level and resilience of sheep to the pathophysiological effects of the infection.

378

In conclusion, genetically controlled markers may exist on ovine lymphocytes which can be used to select disease resistant strains of sheep. Other genetic factors which control the homeostatic mechanisms of sheep may independently influence the resilience of sheep to overcome the harmful effects of infection and m a y therefore provide a useful adjunct to such selection programmes. Similar experiments we have done on cattle infected with the tick Boophilus microplus G.G. Riffkin, C. Dobson and D.E. Moorhouse, unpublished) indicate that resistance to this infection may also be related to pre-infection l y m p h o c y t e responsiveness to the antigens of the tick. ACKNOWLEDGEMENTS

G G R was a recipient of an award under the Commonwealth Scholarship and Fellowship Plan. This work was supported b y a grant from the Australian Research Grants Committee, grant No. DI 68/16765. We thank Mr. J.J. Mines, Mrs. M.E. Owen and Mrs. D. Rogers for their excellent technical assistance, Dr. T.K. Bell of the Department of Physiology for haemoglobin typing of the sheep used in this study and Mr. R. Sheard for his assistance with the mathematics.

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